Early Complement Activation and Decreased Levels Of

Early Complement Activation and Decreased Levels of Glycosylphosphatidylinositol-Anchored Complement Inhibitors in Human and Experimental Diabetic Retinopathy Jing Zhang, Chiara Gerhardinger, and Mara Lorenzi Diabetic retinal microangiopathy is characterized by operative in causing retinal vascular damage in diabetes increased permeability, leukostasis, microthrombosis, and how they are triggered are the objects of ongoing and apoptosis of capillary cells, all of which could be investigation. caused or compounded by activation of complement. In Activation of the complement cascade can both com- this study, we observed deposition of C5b-9, the termi- pound and initiate thrombosis, leukostasis, and apoptosis. nal product of complement activation, in the wall of On the one hand, microthrombi and leukostasis can cause ؎ retinal vessels of human eye donors with 9 3 years of ischemia-reperfusion, which activates complement via the type 2 diabetes, but not in the vessels of age-matched nondiabetic donors. C5b-9 often colocalized with von classical pathway (4), and apoptotic endothelial cells can Willebrand factor in luminal endothelium. C1q and C4, trigger the alternative pathway (5). On the other hand, the complement components unique to the classical complement activation is procoagulant, proinflammatory, pathway, were not detected in the diabetic retinas, and proapoptotic (6), and primary activation of comple- suggesting that C5b-9 was generated via the alternative ment on autologous cells can occur because of insufficient pathway, the spontaneous activation of which is regu- activity of plasma and/or cell surface inhibitors. Sponta- lated by complement inhibitors. The diabetic donors neous cleavage of plasma C3 generates C3b, which at- showed a prominent reduction in the retinal levels of taches covalently to the endothelial cell surface through CD55 and CD59, the two complement inhibitors linked to the plasma membrane by glycosylphosphatidylinosi- its reactive thioester group (5). Additional activation steps tol anchors, but not in the levels of transmembrane are normally prevented by complement inhibitors (7). CD46. Similar complement activation in retinal vessels Among these are the cell surface membrane co-factor and selective reduction in the levels of retinal CD55 and protein (MCP; CD46) and decay accelerating factor (DAF; CD59 were observed in rats with a 10-week duration of CD55), which act to restrict the activity of the C3/C5 streptozotocin-induced diabetes. Thus, diabetes causes convertase enzymes, and protectin (CD59), which inhibits defective regulation of complement inhibitors and com- the final step in the assembly of the membrane-attack plement activation that precede most other manifesta- complex (MAC), the terminal and potentially cytotoxic tions of diabetic retinal microangiopathy. These are novel clues for probing how diabetes affects and dam- product of complement activation. Targeted mutation of ages vascular cells. Diabetes 51:3499–3504, 2002 the gene encoding Crry, the rodent complement inhibitor that exhibits both MCP- and DAF-like activity, causes embryonic lethality because of complement activation at the fetomaternal interface (8). Inherited deficiency of he metabolic abnormalities of diabetes cause CD59 in humans causes paroxysmal nocturnal hemoglo- damage to blood vessels throughout the body. binuria (9), and experimental neutralization of CD59 ac- Damage to the retinal capillaries is manifested tivity in rats augments complement-mediated glomerular Twith abnormal permeability that can lead to damage (7). macular edema and with occlusion and obliteration that Glomerular structures (10) and endoneurial micro- can engender retinal ischemia and unregulated angiogen- vessels (11) of patients with diabetes show signs of esis. Underlying processes documented to date are micro- complement activation. Decreased availability or effec- thrombosis (1), leukostasis (2), and accelerated apoptosis tiveness of complement inhibitors in diabetes is suggested of vascular cells (3). Whether these are the only processes by the findings that high glucose in vitro selectively decreases on the endothelial cell surface the expression of CD55 and CD59 (12), the two inhibitors that are glyco- From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. sylphosphatidylinositol (GPI)-anchored membrane pro- Address correspondence and reprint requests to Mara Lorenzi, Schepens teins (7), and that CD59 undergoes nonenzymatic Eye Research Institute, 20 Staniford St., Boston, MA 02114. E-mail: [email protected]. glycation that hinders its complement-inhibitory function Received for publication 26 June 2002 and accepted in revised form 16 (13). In this work, we investigated whether complement August 2002. activation is a feature of human nonproliferative diabetic DAF, decay accelerating factor; GPI, glycosylphosphatidylinositol; mAb, monoclonal antibodies; MAC, membrane attack complex; MCP, membrane retinopathy and is associated with changes in inhibitory co-factor protein; RBC, red blood cell; vWf, von Willebrand factor. molecules. We extended the study to an animal model of DIABETES, VOL. 51, DECEMBER 2002 3499 COMPLEMENT IN THE DIABETIC RETINA TABLE 1 Characteristics of eye donors and specimens Eyes: time to Eyes: time to Age Sex Diabetes duration enucleation processing Groups (years) (M/F) (years) (h) (h)* Immunohistochemical studies Nondiabetic 66 Ϯ 6 8/6 4 Ϯ 113Ϯ 4 Diabetic 67 Ϯ 6 6/7 9 Ϯ 33Ϯ 112Ϯ 5 Protein studies Nondiabetic 64 Ϯ 7 14/1 3 Ϯ 131Ϯ 6 Diabetic 66 Ϯ 7 11/6 9 Ϯ 34Ϯ 332Ϯ 7 Data are means Ϯ 1 SD. *The time elapsed from death to retina processing was longer for the eyes used for protein isolation (fresh samples) than for the eyes used in the morphological studies because the latter were fixed by the eye banks before shipment. nonproliferative diabetic retinopathy to test the universal- mAb MEM-43 (1 ␮g/ml) and goat polyclonal Ab N-20 (1:1,000; Santa Cruz). ity and timing of abnormalities. Neuron-specific enolase was detected with mAb BBS/NC/VI-H14 (1 ␮g/ml; Dako). In the rat retinas, CD46, CD55, and CD59 were detected with rabbit polyclonal antibody H-294 (1:1,000), H319 (1:200), and R-79 (1:1,000), respec- RESEARCH DESIGN AND METHODS tively (all from Santa Cruz), and ␤-actin with mouse mAb AC-15 (1:200,000; Eye donors and specimens. Human postmortem eyes were obtained from Sigma, St. Louis, MO). certified eye banks through the National Disease Research Interchange. To determine whether the antibodies chosen could detect CD59 quantita- Criteria for inclusion in the study were age Ͻ75 years, diabetes duration Ͻ15 tively in diabetic samples irrespective of glycation, we tested the antibodies years to address mostly nonproliferative retinopathy, and the fewest possible against red blood cells (RBCs) CD59 glycated in vitro. Normal human RBCs chronic pathologies other than diabetes. Criteria for exclusion were retinal, were incubated in standard storage medium containing 55 mmol/l glucose or hematological, and inflammatory diseases; uremia; chemotherapy; and use of in storage medium containing 150 mmol/l glucose for 40 h at 30°C, following life support measures. The causes of death were almost exclusively cardio- a protocol previously described (17). Glycation of hemoglobin was measured vascular in both groups, and hypertension was reported in one-third of donors with the Glyc-Affin GHb Assay (Perkin Elmer Wallac, Norton, OH). Equal in each group. The eyes used for immunohistochemical studies were from 13 amounts (200 ␮g) of solubilized protein from the high- and control-glucose diabetic and 14 nondiabetic donors (Table 1). The eyes were fixed in 10% RBC were incubated with mouse mAb BRA-10G anti-human CD59 (20 ␮g/ml; buffered formalin by the eye banks; one retina from each donor was Ancell, Bayport, MN) at 4°C overnight. The immunocomplexes were precipi- cryopreserved and embedded in OCT compound for sectioning, and the other tated by the addition of protein-G Sepharose (Sigma) for 2 h, and the resulting was in most cases digested with trypsin to isolate the intact microvascular immunoprecipitates were analyzed by Western blotting (14). CD59 glycation, network (trypsin digests) (3). The retinal trypsin digests of seven donors in known to lead mostly to formation of Amadori product (13), was measured each group had previously been studied for microvascular cell apoptosis and with a polyclonal antibody (provided by N. Taniguchi) that reacts with histological lesions of nonproliferative diabetic retinopathy (3). The eyes used ⑀-(1-deoxyhexitolyl)-lysine, the reduced form of the Amadori product (18). for protein isolation were from 17 diabetic and 15 nondiabetic donors Hence, the blots were treated with NaHBH4 (50 ␮mol/l in PBS) for4hatroom (Table 1). temperature, followed by a 15-min wash in PBS acidified to pH 5.0 with acetic Animals and specimens. Six-week-old male Sprague-Dawley rats (Taconic acid to stop the reaction. After additional PBS washes, the membrane was Farms, Germantown, NY) were assigned randomly to a diabetic and a control blocked and reacted with the anti–hexitol-lysine antibody (1:1,000). The group. Diabetes was induced by intravenous administration of streptozotocin amount of immunoprecipitated CD59 was measured in companion blots (55 mg/kg body wt, dissolved in citrate buffer pH 4.5). The care of the diabetic probed with the anti-CD59 mAb MEM-43 or polyclonal antibody N-20. Similar rats and insulin treatment to prevent weight loss were

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